1a. Objectives (from AD-416)
Isolate and identify genes involved in the biosynthesis of phytotoxins involved in pathogenicity of fungal pathogen in cotton.
1b. Approach (from AD-416)
Phytotoxins that may be involved in the pathogenicity of fungal wilt pathogens that affect cotton yield and quality will be investigated. Specifically, the genes that control their biosynthesis or expression will be identified. The fungal compounds currently being considered for investigation are expected to be derived via a polyketide synthase. This will be confirmed using 13C-labelled feeding studies using singly and doubly labelled acetate under conditions that foster rapid production of the metabolites under investigation. Methods that suppress biosynthesis will also be determined. mRNA will be extracted from mycelium and a pair of degenerate primers corresponding to conserved regions of the ketoacyl synthases (KS) domain of the PKS genes will be used to amplify the KS domain of the PKS genes form the extracted mRNA. The expected base pair will be cloned and transformed into E. coli cells. Plasmids will be isolated from the transformed cells and the inserts sequences will be identified. Once PKS genes are substantiated based on the sequence homology to known fungal KS domain sequences, a pair of primers will be synthesized based on the cloned sequences. mRNA isolated at various incubation periods under both metabolite suppression and induction conditions will be used as a template in RT-PCR to analyze the expression profile of the PKS genes corresponding to the cloned fragments. This will enable us to identify the clones involved in metabolite biosynthesis.
3. Progress Report
The goal of this project is to establish how certain naturally occurring chemicals found in fungi affect pathogenicity of these fungi to cotton. Project work in FY 2011 studied the biosynthesis of the phytotoxin fusaric acid, which is produced by Fusarium oxysporum f. sp. vasinfectum. Using carbon-13 labeled compounds, the work established that the incorporation of labeled substrates is consistent with the biosynthesis of fusaric acid from three acetate units at C5-C6, C7-C8, and C9-C10, with the remaining carbons being derived from aspartate via oxaloacetate and the tricarboxylic acid cycle. The oxaloacetate originates in part by transamination of aspartate, but most of the oxaloacetate is derived by deamination of aspartate to fumarate by aspartase. The nitrogen from glutamine is more readily incorporated into fusaric acid than from aspartate. As the work progresses, new information will be developed that will advance our understanding of the pathogenicity of these new types of disease-causing fungi. The work will also provide foundational information to support efforts aimed at developing new disease-resistant cotton cultivars for productive use by U.S. farmers. The ADODR of this project and the cooperator are located in close physical proximity, and are in regular contact with each other for discussions on the direction and progress of the project.